Rab GTPases are essential regulators of diverse cellular processes including membrane trafficking, cell growth, organelle biogenesis, membrane remodeling, signaling transduction, and development. Rab GTPases as well as their regulatory factors and effectors have been implicated in genetically linked disorders, complex disease states such as type II diabetes and cancer, and are targets of virulence factors from pathogenic organisms. Many Rab GTPase have spatially and temporally overlapping distributions within the dynamic and highly interconnected network of tubular-vesicular organelles that comprise the biosynthetic, endocytic, and recycling systems. The functions of Rab GTPases are further coordinated through multivalent proteins and complexes with two or more distinct Rab GTPase binding domains, each of which has a unique specificity profile for subsets of Rab GTPases. With 60 distinct proteins encoded in the human genome, Rab GTPases represent the largest and most complex branch of the Ras superfamily. Characterizing interactions with structurally diverse effectors and regulatory factors, determining specificities, and deciphering the elaborate encoding of recognition determinants represents a critically important challenge. During the previous funding period, we developed, optimized, and successfully tested a multidisciplinary strategy for solving the recognition problem at the level of the Rab GTPase family. The long term objectives of this renewal application are to: i) generalize this approach to as many effectors, regulatory factors, and accessory proteins as possible; and ii) extend the methodology to incorporate identification of interaction partners for candidate Rab interacting proteins. To achieve these objectives, we will: (Aim 1) quantitatively profile the specificity of effectors and regulatory factors for the Rab GTPase family; (Aim 2) investigate the underlying structural bases; (Aim 3) identify the major determinants of the observed specificity. This combination of experimental strategies will provide critical information on the specificity profiles, structural bases, and sequence determinants underlying the interaction of Rab GTPases with effectors, regulatory factors, and accessory proteins. Relevance to Public Health These studies will reveal novel insights into the mechanisms for regulation of membrane trafficking, cell growth, and metabolism. The resulting information may prove useful in the design of mechanism based therapeutics for treatment of cancer and type II diabetes.